Water-soluble polymer-enzyme products

ABSTRACT

Polymer-enzyme products wherein the enzyme is covalently bound to the polymer chain, the polymer-enzyme product being water soluble. The products have wide applicability as stable, longacting, enzymatic materials, having same general type of activity as parent enzyme, but with different pH optimum activity and range of applicability, and are stable, substantially colorless and odorless, long acting, and remarkably less subject to autogenous deterioration or destruction by other enzymes.

United States Patent Bernard S. Wildi Kirkwood;

Thomas L. Westman, St. Louis, both of Mo. 763,343

Sept. 27, 1968 Dec. 7, 197 1 Monsanto Company St. Louis, Mo.

[72] Inventors [21 Appl. No. [22] Filed [45] Patented [73] Assignee [54]WATER-SOLUBLE POLYMER-ENZYME PRODUCTS 19 Claims, No Drawings [52] U.S.Cl 195/63, l95/DlG. 11, 195/68 [51] lnt.Cl Cl2k 1/00 [50] Field ofSearch 195/63, 63 P, 66, 68

[56] References Cited UNITED STATES PATENTS 3,436,309 4/1969 Ottinger eta1.. 195/63 X 3,507,750 4/1970 Murray et al. 195/66 OTHER REFERENCESMitz, et al., Nature, Feb. 1961 Vol. 189 (pgs. 576- 577).

Goldstein, et al., Biochemistry, Dec. 1964 Vol. 3, No. 12 (pgs.1913-1914).

Hornby, et al., Biochemical Journal, 1966, Vol. 98 (pgs. 420-425).

Proceedings of the Biochemical Society, Biochemical Journal, Mar. 1968,Vol. 107 (pgs. 2?, 3p, and 5p).

Primary Examiner-Lionel M. Shapiro Assistant ExaminerD. M. NaffAttorneys-Gordon W. Hueschen, Hueschen and Kurlandsky and John D. UphamABSTRACT: Polymer-enzyme products wherein the enzyme is covalently boundto the polymer chain, the polymer-enzyme product being water soluble.The products have wide applicability as stable, long-acting, enzymaticmaterials, having same general type of activity as parent enzyme, butwith different pH optimum activity and range of applicability, and arestable, substantially colorless and odorless, long acting, andremarkably less subject to autogenous deterioration or destruction byother enzymes.

1 WATER-SOLUBLE POLYMER-ENZYME PRODUCTS BACKGROUND OF INVENTION 1. Fieldof Invention Polymer-enzyme products; water soluble.

2. Prior Art It is well known that native enzymes often displayinstability in solution and that such instability results from autolysisin the case of proteolytic enzymes or, for other types of enzymes,accompanying proteases cause inactivation by digestion of said enzymes.Similarly, native enzymes may become inactivated due to denaturationcaused by heat, the pH of the solution, and/or other well-documentedphenomena.

Examples are also to be found in the literature of simple chemicallymodified enzymes (e.g., succinylated, nitrated, etc.) which haveenhanced or modified enzymatic properties. In general, however, theseenzyme derivatives often do not display enhanced solution stabilitycompared to the native enzyme. In addition, an attendant problemassociated with such enzyme derivatives is the difficulty in separatingthese derivatives from the native enzyme.

Some insoluble polymeric derivatives of enzymes have been shown to haveenhanced solution stability as compared to the native enzymes or theirsimple chemical derivatives. Moreover, isolation of insolublepolymer-enzyme products is readily achieved as via centrifugation andremoval of unbound enzyme is achieved by washing of the insolublepolymer-enzyme products.

Whereas in certain cases it may be desirable to utilize a stable,insoluble polymeric derivative of an enzyme, such utilization involvescontacting a substrate solution with the solid enzyme derivative, or asolid substrate with the solid enzyme derivative. It is well known thatsuch heterogeneous phase reactions are less efficient due to poorcontact of substrate with the enzyme catalyst. This particularlyapparent for biochemical reactions where the substrate, such as aprotein,

lipid or polysaccharide, is often large and the enzyme is also large aswell. Moreover, such insoluble polymer enzyme derivatives employ amarked degree of cross linking to effect insolubility. Such crosslinking can therefore often cause entrapment of the enzyme within thethree-dimensional network with resulting inaccessibility of the enzymeto the substrate, thus reducing the entrapped enzyme to an ineffectivecatalyst. A solution to this problem of more convenient contact would behighly desirable.

As already stated, insoluble polymer-enzyme derivatives are known.Insoluble polymer-enzyme products reported have been characterized by avery desirable pH shift of optimum enzymatic activity, which in effectprovides a new enzyme, operative in the same or substantially the sameway, but under different conditions than, the native enzyme. Frequently,these insoluble products have a diminished, reduced, or different typeor extent of enzymatic activity than the parent enzyme, beingcharacterized by a different bond-splitting or hydrolyzing capacity thanthe parent enzyme. Such changed characteristics, in effect giving riseto a new enzyme, would be highly desirable in a soluble polymer-enzymesystem. However, the researchers who have reported the insolubleenzymepolymer products of these remarkably differing properties, havenot reported corresponding water-soluble enzymepolymer products.

The only case of a soluble enzyme-polymer product known to us hasreportedly provided products having enzymatic activity, in fact greatereven than the enzyme from which they were derived. However, theseauthors also reported that there was no change in the pH activitycharacteristics of their particular trypsin and chymotrypsin polymerproducts. [Mitz et al., Nature 189, 576 (1961)]. In theory, thisreported finding could be explained by more complete substrateaccessibility to the polymer-enzyme due to its soluble form but,whatever the explanation, this reported result was disappointing fromthe standpoint of effectively providing new enzymatic products which areconstructively tantamount to new enzymes, having their greatest activityin a different range than the parent, and thus giving rise to differentapplications for which the parent is not ideally, if at all, suited.Thus, to the present, no soluble polymer-enzyme products have beenavailable which differ from the parent enzyme in the importantcharacteristic of pH optimum activity, if in fact any solublepolymer-enzyme product has been available at all. We have unpredictablybeen able, according to the present invention, to provide the same. Suchprovision is an unobvious, unpredictable, and important advance in theart.

SUMMARY OF THE INVENTION The present invention provides novelwater-soluble products in which an enzyme is covalently bonded to aselected polymer and a process for their production. These novelpolymer-enzyme products have valuable and unpredictable properties. Whytheir properties differ from the properties reported by Mitz et al. isnot presently known, and perhaps may never be ascertained, but sufficeit to say that the cited publication leads directly away from arecognition that any soluble polymer-enzyme products have or may havethe highly desirable pH activity shift, as we have found to characterizeour different trypsin and chymotrypsin-polymeric products, in directcontrast to Mitz et al. who state that there was no shift in the pHoptimum for their corresponding but still importantly difi'erent trypsinand chymotrypsin-polymeric products.

Unexpectedly, it has now been found that soluble polymer enzyme productscan be prepared which possess the remarkably modified enzyme propertiesof the insoluble polymeric derivatives. The soluble chymotrypsin andtrypsin polymer products of the invention have been found to have a pHactivity profile strikingly different from the native enzyme. This isentirely unexpected in view of Mitz et al. and even more unexpectedsince the substrate should have more complete accessibility, to theattached enzyme which is now not associated with a cross-linked system,as is characteristic of insolubles.

Also surprisingly, the stability of these soluble polymer-enzymederivatives is still found to be increased relative to the nativeenzyme, and this increased stability may be the result of severalpossible factors but it cannot be readily explained at this time. At anyrate, these soluble products are remarkably stable against autolysis anddenaturation, even in solution.

Moreover, in the case of the simple chemically modified enzymes, asmentioned above, these cannot be separated readily from native enzymeexcept with extreme difficulty and usually only on a small scale. Atotally unexpected discovery was made that soluble polymer-enzymeproducts can be separated from unattached enzyme by chromatography oncross-linked dextrans (Sephadex Pharmacia Co., Uppsala, Sweden). it wasdiscovered that hydrolyzed polymer (e.g., EMA) and the solublepolymer-enzyme product are eluted in the void volumes of such columnsregardless of the exclusion limits of the Sephadex (up to and includingSephadex G-200, exclusion limits approximately 150,000 molecularweight). Furthermore, it has been discovered that Sephadexchromatography of mixtures of soluble polymeric derivatives of enzymeswith the native enzyme and also mixtures of polymer, e.g., hydrolyzedEMA, and enzymes in general are readily separated from one another bySephadex chromatography providing the enzyme is not of a molecularweight so large as to be eluted in the void volume of such columns.

That this separation technique is valid is shown by the fact thatchromatographically purified soluble polymer-enzyme products do notmigrate upon disk gel electrophoresis whereas enzymes in the presence ofpolymers, e.g., hydrolyzed EMA, do migrate normally. In addition, it hasbeen shown that enzyme and hydrolyzed polymer, e.g., hydrolyzed EMA, areseparated by Sephadex chromatography as evidences by low optical densityof void volume eluates as measured at 280 mu. Similarly, biologicalactivity and nitrogen content of void hydrolyzed EMA, mixtures low whenenzyme-polymer, e.g., are chromatographed on Sephadex. On the otherhand, the soluble polymer-enzyme derivatives possess biologicalactivity. This method therefore constitutes a process whereby solublepolymer-enzyme products can be separated from contaminating and unboundenzyme due to their unique physical properties.

As mentioned above, the soluble polymer-enzyme products therefore can besaid to have the desirable property of solubility such as of the nativeenzyme, facilitating desirable substrate contact, and concomitantly alsopossess the desirable properties of enhanced stability and modifiedbiological behavior such as characterize the insoluble polymer-enzymederivatives. I

Such soluble polymer-enzyme products have advantages uniquely differentfrom either native enzymes or their insoluble polymeric derivatives. Forexample, such soluble derivatives may be used to act upon biological andsynthetic substrates based fundamentally upon the characteristicbiological action of the attached enzyme but within their optimallyactive pH range. Subsequently such soluble derivatives may be separatedfrom the treated substrate by Sephadex chromatography as describedabove. The soluble polymeric derivative may then be reisolated andreused.

Alternatively, the soluble enzyme-polymer product may be contained in achamber to which is attached a porous membrane as one wall thereof. Sucha porous membrane possesses the ability to allow passage of lowmolecular weight substances but not those of high molecular weight, suchas the soluble polymer-enzyme derivative. Hence, the solubleenzyme-polymer product is used to treat substrates such that, aftertreatment, the products from digestion of the substrate are separatedfrom the soluble enzyme-polymer product by passage through thesize-limiting porous membrane whereas the soluble enzyme-polymer productremains behind for subsequent isolation or reuse.

One example of this latter type of processing involves SEMAT (SolubleEMA-Trypsin) digestion of RIBOX (oxidized ribonuclease) according to C.H. W. Hirs, J. Biol. Chem. 219, 61 l (1956). The reaction is allowed toproceed to completion in a Diaplex Ultrafil Model 400 Cell (AmiconCorporation, Cambridge, Massachusetts) to which is attached a UM-lDiaflo Membrane (Amicon Corp.), exclusion limits ca. 10,000. After thereaction is completed, the digested RIBOX fragments are separated bypassage through the membrane whereas the SEMAT remains contained in thereaction vessel. Subsequently, the vessel is recharged with RIBOXsolution, the digestion proceeds, and products are isolated as above.Hence, there is provided a method for continuous, semicontinuous, orbatchwise biological processing, isolation of products, and subsequentreuse of the soluble polymer-enzyme derivative.

Another example of a highly advantageous use of soluble polymer-enzymeproducts involves their ease of application in the form of stablesolutions. For example, a soluble cellulase/EMA derivative in solutionform can be sprayed onto cellulose or cellulose-containing material withsubsequent digestion of the cellulose, releasing glucose. Recovery andreuse is possible. Other advantages accruing to the novel solublepolymer-enzyme products of the invention and in their use are toonumerous to mention and will be apparent to one skilled in the art.

volume eluates are OBJECTS The provision of novel water-solublepolymer-enzyme products having any or all of the foregoing enumeratedadvantages or advantageous properties or characteristics, and a processfor the production of such water-soluble polymer-enzyme products, is oneof the objects of the present invention. Other objects will becomeapparent hereinafter and still others will be obvious to one skilled inthe art.

GENERAL DESCRIPTION OF THE INVENTION Definitions EMA is a polymer ofethylene and maleic anhydride. Polymers of this type are of great valueaccording to the present invention.

EMA-type" polymer is defined hereinafter.

EMA-enzyme or EMA/enzyme" is a copolymer of ethylene and maleicanhydride having enzyme covalently bonded thereto. The product is thesame whether the enzyme is reacted directly with an anhydride group ofthe ethylenemaleic anhydride copolymer or with a carboxyl group ofethylene-maleic anhydride copolymer, whether or not using anintermediate activating mechanism for carboxyl groups of the polymer.Anhydride groups not participating in the reaction by which the productis produced in aqueous medium are present in the product as carboxyl orcarboxylate groups. Such nonparticipating groups may, however, beconverted to amide, imide, ester, etc., groups, as can be present inEMA- type polymers, as hereinafter defined.

Water insoluble" means that the product concerned does not dissolve inwater or aqueous solutions, although it may have such characteristics asa high degree of swelling due to water solvation, even to the extent ofexistence in gel form. Water soluble" means not water insoluble, and isfurther defined hereinafter.

The product of the invention is a water-soluble enzymatically activepolymer-enzyme product wherein the enzyme is bound covalently through agroup which is nonessential for enzymatic activity to (a) a polymercomprising chains of carboxylic acid or carboxylic acid anhydride units,said polymer chains being formed by polymerization of polymerizableacids or anhydrides, or (b) a polymer comprising units of carboxylicacid or carboxylic acid anhydride groups separated by carbon chains ofat least one and not more than four carbon atoms, said carbon chainsbeing part of a unit which contains a maximum of 18 carbon atoms, saidpolymer being formed by copolymerizing a polymerizable acid or anhydridewith another copolymerizable monomer, and preferably wherein thestarting acid or anhydride and any additional copolymerizable monomerare unsaturated and such polymerization or copolymerization comprisesaddition-type polymerization or copolymerization involving suchunsaturation.

Process Polymer-enzyme derivatives can be prepared by reacting thecrystalline or crude enzyme or mixture of enzymes with the polymer insolution, resulting in formation of a polymeric product in which theenzyme is covalently bound. Since an anhydride or carboxyl is present inthe polymer, e.g., an EMA- type polymer, covalent bonding of the enzymeto the polymer may be effected directly through reaction of couplingwith an anhydride group or with a carboxyl group using a carboxylactivating agent. The product is the same in both cases. The pH rangefor the reaction depends upon the enzyme employed and its stabilityrange. It is usually about 5 to 9.5, preferably about 6-8, butadjustment must be made for individual cases. Isolation and purificationis generally effected according to normal biochemical procedures, and bythe general procedure of the examples which follow. Since covalentbonding of the enzyme to the polymer is desired, the reaction isordinarily carried out at low temperatures and at relatively neutralpH's, in water or dilute aqueous buffer as solvent.

When carried out in this manner, the results are production of thedesired active polymer-enzyme derivative, but degree of activityimparted to the polymeric product is sometimes lower than desired,possibly due to partial inactivation of the enzyme during the process.Resort may frequently advantageously be had to employment of a mixedsolvent system, using a solvent in which the enzyme is at leastpartially soluble, usually in an amount up to about 50 percent byvolume. Dimethylsulfoxide (DMSO) is especially suitable as solventtogether with water or aqueous buffer solution in a mixed solventsystem. Using such a mixed solvent system, the desired activeenzymepolymer product is ordinarily obtained in higher yields andconversions to desirably active product, and introduction of desirablyhigh amounts of enzyme activity into the polymer molecule is generallyless difficult.

As stated, the polymer in such reaction contains carboxyl or anhydridelinkages, especially where the enzyme contains an amino, hydroxyl(including phenolic hydroxyl), or sulfhydryl group not essential for itsenzymatic activity. The polymer is preferably EMA or an EMA-typepolymer, but it can be any of those types herein disclosed for couplingor reaction with an enzyme, and in any event it is adapted to effectcovalent bonding with the enzyme to produce an enzyme-polymer producteither directly or indirectly using an activating agent. Inasmuch as theenzymatic activity of the starting enzyme is desired to be retained inthe final product, it is of course firstly necessary that bonding of theto the polymer be through a group which will not result in inactivationof an active site in the enzyme molecule. Among the various reactivegroups of enzyme molecules may be mentioned, besides amino andsulfhydryl, also hydroxyl (including phenolic hydroxyl), carboxyl andimidazolyl. Such groups are present in free or unbound form in inactiveportions of enzyme molecules, as in a lysine, cysteine, serine,threonine, histidine, or tyrosine moiety of an enzyme molecule, wherethe particular moiety in question is not considered essential forenzymatic activity, either catalytic in nature or for substrate binding.Therefore, attachment to the polymer molecule is through reaction of thepolymer with such group so as to avoid inactivation of the enzyme duringattachment to the polymer molecule. Generally the linkage is an amide,imide, ester, thioester, or disulfide group, such as formed by thecarboxyl or anhydride with an amine or hydroxyl group in a nonessentialmoiety of the enzyme protein chain. Amides are conveniently formed byreacting pendant amino groups of the enzyme with carboxylic anhydridegroups on the carrier polymer in water, in aqueous buffer media, or inmixed solvents. Amides, imides and esters are readily formed byactivating carboxyl groups of the polymer, and reacting them withrespective hydroxyl, amine or mercaptan groups on the other reactant.Such activation may be effected using various carbodiimides,carbodiimidazoles, Woodwards or Sheehans reagent, or the like, to formhighly active intermediates capable of reacting with groups in theenzyme under mild conditions, the latter favoring retention of enzymaticactivity.

The polymer selected for such reaction can therefore be said to beadapted to couple or react with the enzyme, either directly orindirectly through use of an activating agent, as already indicated, andin any event to effect covalent bonding with the enzyme. The attachmentprocedures given are conducted by techniques adapted to include anyrequisite protection for the enzyme, which may include a reversibleblocking of the enzymatically active site or sites, as for example inthe case of papain, where mercuripapain or zinc papain may be employedas an intermediate for reaction with the polymer in order to effectgreater yields upon attachment, the protecting atoms being removedsubsequent to the attachment reaction. General Procedure for SolublesPreparation In order to achieve high yields of water-solubleenzymepolymer products, it is desirable to avoid cross linking whichresults in insolubilization.

To prepare water-soluble enzyme-polymer derivatives, therefore, thereaction is preferably performed under substantially noncross-linkingconditions. The undesired cross linking can be reduced by performing theattachment reaction in high dilution such that fewer reactions occurbetween several polymer molecules and a single enzyme molecule.Alternatively, high ratios of enzyme to polymer favor reaction ofseveral enzyme molecules with a single polymer molecule. This,therefore, results in an agglomerated enzyme/polymer system whichmaintains the desired soluble properties of the individual enzymemolecules. An additional way of favoring solubles" formation is to runthe reaction at high ionic strength to decrease aggregation of thenative protein. While such procedures as described above are oftendesirable, it is not always necessary to use dilute solutions or highenzyme/polymer ratios to cause formation of soluble enzyme/polymerderivatives.

The term water soluble" means that the product concerned dissolves inwater or aqueous solutions. As usual, how ever, this does not mean thatthe product dissolves completely at all concentrations or at all pHs. Onthe other hand, these water-soluble products are characterized by beingsoluble at a variety of concentrations and pHs, and they are generallysoluble at pl-ls of 7 or greater.

In their soluble form, the polymer-enzyme products of the invention arecharacterized by fundamentally the same enzymatic activity as the parentnative enzyme, but have all of the advantages which are attendant uponapplicability in solution or suspension form together with increasedstability and prolonged activity. In addition, because of theirpolymeric form, even though soluble, the polymer-enzyme products of theinvention are separable from native enzyme or substrates, as well asimpurities and coloring matter of an undesired nature, by nonnalseparation procedures such as centrifugation, electrophoresis, orchromatography.

POLYMERIC REACTANT In its broadest context, the polymer to which theenzyme is to be coupled according to one or more aspects of theinvention contains carboxyl or anhydride linkages, especially where theenzyme contains an amino, hydroxyl, or sulfhydryl group not essentialfor its enzymatic activity. The polymer may be EMA or an EMA-typepolymer, or be any of those types disclosed herein for coupling orreaction with an enzyme, and in any event it is adapted to couple orreact with the enzyme to effect covalent bonding and production of thedesired soluble enzyme-polymer product. 4

Since covalent bonding is desired, it is understood that the carrierpolymer is tailored to contain at least one reactive site for eachpolymer molecule with which the enzyme can react, either directly orindirectly, to produce a covalent bond. According to the instantinvention, this reactive site (or sites) is preferably a carboxyl orcarboxylic anhydride group.

The polymeric reactant, according to the invention, may be definedbroadly as follows: (a) a polymer comprising chains of carboxylic acidor carboxylic acid anhydride units, said polymer being formed bypolymerization of polymerizable acids or anhydrides, or (b) a polymercomprising units of carboxylic acid or carboxylic acid anhydride groupsseparated by carbon chains of at least one and not more than four carbonatoms, said carbon chains being part of a unit which contains a maximumof 18 carbon atoms, said polymer being formed by copolymerizing apolymerizable acid or anhydride with another copolymerizable monomer,and preferably wherein the starting acid or anhydride and any additionalcopolymerizable monomer are unsaturated and such polymerization orcopolymerization comprises addition type polymerization orcopolymerization involving such unsaturation.

Among the polymers suitable for the practice of the instant invention,polymeric polyelectrolytes having units of the formula wherein: R, and Rare selected from the group consisting of hydrogen, halogen (preferablychlorine), alkyl of one to four carbon atoms (preferably methyl), cyano,phenyl, or mixtures thereof; provided that not more than one of R A andR n is phenyl; Z is a bivalent radical (preferably alkylene,phenylalkylene, lower-alkoxyalkylene, and lower-aliphaticacyloxyalkylene) comprising a carbon chain having one to four carbonatoms, inclusive, said carbon chain being a part of a unit whichcontains one to 18 carbon atoms, inclusive, q is zero or one, X and Yare selected from hydroxy, alkali metal, OR, ONH ONHR 'ONH,R,, 0NH R,NRR', and (Q),,--W (NR'R') (Q),,W-(OH),, wherein at is l to 4 and p iszero or 1, wherein R is selected from the group consisting of alkyl,phenylalkyl, or phenyl, in each case of one to 18 carbon atoms, whereinR is H or R, wherein Q is oxygen or NR'-, and wherein W is a bivalentradical preferably selected from lower-alkylene, phenyl, phenylalkyl,pehnylalkyl-phenyl, and alkylphenylalkyl having up to 20 carbon atoms, Xand Y taken together can be an oxygen atom, and at least one of X and Ybeing hydroxyl or X and Y together constituting an oxygen atom, arepreferred. Many of these polymers are commercially available and othersare simple derivatives of commercially available products, which can bereadily prepared either prior to or simultaneously with the enzymecoupling reaction, or produced as a minor modification of the basicpolymer after coupling. Such polymers containing the above-describedEMA-type units are hereinafter referred to as an EMA-type polymer.

Since enzyme molecules have an extremely high molecular weight, even ifthe polymeric units exemplified as usable for attachment of the enzymeoccurs only once in a polymer chain, for example, once in every severalhundred units, reaction of the enzyme with this unit will result in anenzymepolymer product having substantial enzymatic activity and onewherein the enzyme moiety constitutes a substantial portion of themolecular weight of the polymeric enzyme product. If more than one ofthe exemplified units is present, multiple attachments can be achievedwith increased enzymatic activity of the product. As pointed outhereinafter, preferably the units of the formula given are recurring, nbeing at least 8. When the units are recurring, the symbols in thevarious recurring units do not necessarily stand for the same thing inall of the recurring units. Moreover, where the units are recurring,some of the X and Y groups may have meanings besides hydroxy or oxygen.For example, some, but not all, of them may be present in the form ofimide groups, that is, groups in which X and Y together are NR orN-W(NR'R), wherein R, W and R have the values previously assigned.

A preferred type of polymeric material useful in the practice of theinvention is the polymer of an olefinically unsaturated polycarboxylicacid or derivative with itself or in approximately equimolar proportionswith at least one other monomer copolymerizable therewith. Thepolycarboxylic acid derivative can be of the nonvicinal type, includingacrylic acid, acrylic anhydride, methacrylic acid, crotonic acid ortheir respective derivatives, includingpartial salts, amides and estersor of the vicinal type, including maleic, itaconic, citraconic, a,a-dimethyl maleic, a-butyl maleic, a-phenyl maleic, fumaric, aconitic,a-chloromaleic, a-bromomaleic, acyanomaleic acids including theirpartial salts, amides and esters. Anhydrides of any of the foregoingacids are advantageously employed.

Comonomers suitable for use with the above functional monomers includea-olefins such as ethylene, propylene, isobutylene, lor 2-butene,l-hexene, l-octene, l-decene, 1- dodecene, l-octadecene, and other vinylmonomers such as styrene, a-methyl styrene, vinyl toluene, vinylacetate, vinyl amine, vinyl chloride, vinyl forrnate, vinyl propionate,vinyl alkyl ethers, e.g., methylvinylether, alkyl acrylates, alkylmethacrylates, acrylamides and alkylacrylamides, or mixtures of thesemonomers. Reactivity of some functional groups in the copolymersresulting from some of these monomers permits formation of other usefulfunctional groups in the formed copolymer, including hydroxy, lactone,amine and lactam groups.

Any of the said polybasic acid derivatives may be copolymerized with anyof the other monomers described above, and any other monomer which formsa copolymer with dibasic acid derivatives. The polybasic acidderivatives can be copolymers with a plurality of comonomers, in whichcase the total amount of the comonomers will preferably be aboutequimolar with respect to the polybasic acid derivatives. Although thesecopolymers can be prepared by direct polymerization of the variousmonomers, frequently they are more easily prepared by an afterreactionmodification of an existing copolymer.

Copolymers of anhydrides and another monomer can be converted tocarboxyl-containing copolymers by reaction with water, and to ammonium,alkali and alkaline earth metal and alkylamine salts thereof by reactionwith alkali metal compounds, alkaline earth metal compounds, amines, orammonia, either prior to, during, or subsequent to enzyme attachment,etc. Other suitable derivatives of the above polymers include thepartialalkyl or other esters and partial amides, alkyl amides, dialkylamides, phenylalkyl amides or phenyl amides prepared by reactingcarboxyl groups on the polymer chain with the selected amines or alkylor phenylalkyl alcohol as well as amino esters, amino amides, hydroxyamides and hydroxy esters, wherein the functional groups are separatedby lower-alkylene, phenyl, phenylalkyl, phenylalkylphenyl, oralkylphenylalkyl, which are prepared in the same manner in each casewith due consideration of preservation of enzyme attachment sites aspreviously stated. Other aryl groups may be present in place of phenylgroups. Particularly useful derivatives are those in which negativelycharged carboxyl groups are partially replaced with amine or amine saltgroups. These are formed by reaction with said carboxyls with polyaminessuch as dimethylaminopropylamine or dial kylaminoalcohols such asdimethylaminoethanol, the former forming an amide linkage with thepolymer and the latter an ester linkage. Suitable selection of the abovederivatives permit control of several parameters of performance for theenzyme-polymer product of the invention.

Representative dibasic acid or anhydride-olefin polymers, especiallymaleic acid or anhydride-olefin polymers, of the foregoing type (EMAtype) are known, for example, from US. Pat. Nos. 2,378,629, 2,396,785,3,157,595 and 3,340,680. Generally, the copolymers are prepared byreacting ethylene or other unsaturated monomer or mixtures thereof, aspreviously described, with the acid anhydride in the presence of aperoxide catalyst in an aliphatic or aromatic hydrocarbon solvent forthe monomers but nonsolvent for the interpolymer formed. Suitablesolvents include benzene, toluene, xylene, chlorinated benzene and thelike. While benzoyl peroxide is usually the preferred catalyst, otherperox ides such as acetyl peroxide, butyryl peroxide, ditertiary butylperoxide, lauroyl peroxide and the like, or any of the numerous azocatalysts, are satisfactory since they are soluble in organic solvents.The copolymer preferably contains substantially equimolar quantities ofthe olefin residue and the anhydride residue. Generally, it will have adegree of polymerization of eight to 10,000, preferably about to 5,000,and a molecular weight of about 1,000 to 1,000,000, preferably about10,000 to 500,000. The properties of the polymer, such as molecularweight, for example, are regulated by proper choice of the catalyst andcontrol of one or more of the variables such as ratio of reactants,temperature, and catalyst concentration or the addition of regulatingchain transfer agents, such as diisopropyl benzene, propionic acid,alkyl aldehydes, or the like. The product is obtained in solid form andis recovered by filtration, centrifugation or the like. Removal of anyresidual or adherent solvent can be effected by evaporation usingmoderate heating. Numerous of these polymers are commercially available.Particularly valuable copolymers are those derived from ethylene andmaleic anhydride in approximately equimolar proportions. The product iscommercially available.

The maleic anhydride copolymers thus obtained have repeating anhydridelinkages in the molecule, which are readily hydrolyzed by water to yieldthe acid form of the copolymer, rate of hydrolysis being proportional totemperature. In view of the fact that the coupling reactions of thepresent invention are carried out in aqueous solutions or suspensions,or using water-solvent mixtures, the product of the covalent bonding(coupling) of the enzyme to EMA has carboxyl or carboxylate groupsattached to its chains adjacent the coupled enzyme instead of anhydridegroups, due to hydrolysis of the anhydride groups, which do not reactwith the enzymes during the coupling reaction. The same is true ofnonreacting anhydride groups present in other polymers, such as EMA-typepolymers, which hydrolyze to carboxyl or carboxylate groups during thecoupling reaction.

ENZYMES The enzyme starting material may be obtained from any suitablesource. It may comprise one or more of neutral and alkaline proteases,papain, aclepain, bromelin, bromelain, trypsin, chymotrypsin or thelike. It may comprise an 7.25% but a lipase may be used instead of or inaddition to the amylase. A carbohydrase, esterase, nuclease, or othertype of hydrolase may be a starting enzyme reactant. A hydrase,oxidoreductase, or demolase may also be employed, or a transferase orisomerase, depending upon the ultimate activity and applicationintended. At any rate, whatever enzyme or enzyme mixture is employed asstarting material according to the invention, it or they will becovalently bound into the watersoluble polymer-enzyme molecule.

A great many enzymes are known and are suitable for incorporation intothe water-soluble polymer-enzyme products of the invention. Numerousstarting enzymes are available commercially, being obtained from variousanimal, vegetable, and microbial sources. Many enzymes are obtained bymicrobial fermentation, e.g., production of enzymes by bacteria, usingwell-known fermentation methods such as those generally described inKirk-Othmer, Encyclopedia of Chemical Technology 8, 173-204.

The exact activity of the enzyme or enzymes employed as startingmaterial in the invention is not critical, providing only that thestarting enzyme has the desired activity suitable for the ultimatelyintended use of the product. Various analytical methods are available todetermine the activity of enzymes and enzymatically active materials,for example, the protease activity of proteolytic enzymes can bedetermined by wellknown casein digestion methods. According to suchtests, a protease catalyzes the hydrolysis of casein for a certainperiod of time and temperature and a certain pH; the reaction is stoppedby the addition of trichloroacetic acid, and the solution is filtered.The color of the filtrate is developed by Folin phenol reagent, and thelevel of enzyme activity is measured spectrophotometrically in units ofcasein tyrosine. This method is more fully described in the Journal ofGeneral Physiology 30, 291 (1947) and in Methods of Enzymology 2, 33 byAcademic Press, NY. (1955). Amylase activity is generally determined bythe well-known dinitrosalicylic acid method of Bernfeld. Other tests areset forth hereinafter.

A particularly effective source of mixed enzymes which can be used asstarting material in the present invention is a mutated Bacillussubtilis organism. The process for producing this organism and enzymestherefrom is described in U.S. Pat. No. 3,031,380. A culture of thisBacillus subtilis (strain AM) organism has been deposited with the U.S.Department of Agriculture, Agricultural Research Service, NorthernUtilization Research and Development Division, 1815 North UniversityStreet, Peoria, 111. 61604, and has been assigned No. NRRL B-34l l. Theenzymatically active material produced by this organism has been foundgenerally to consist of two proteases, approximately 65-75 percentneutral protease (activity at pH of 7.07.5) and about 25-35 percentalkaline protease (activity at pH of 9 to 10). A significant amount ofamylase is also present. There are generally about 700,000 to about1,200,000 units of neutral protease activity per gram of isolated solidsand about 250,000 to about 400,000 units of alkaline protease activityper gram as determined by Ansons variation of the Kunitz Caseindigestion method. There are generally about 300,000 to 350,000 units ofamylase activity per gram as determined by the Bernfeld method. Aspointed out in the cited patent, the relative proportions of protease toamylase will vary depending on the exact conditions of growth of themicro-organism, but we have found that the neutral and alkaline proteaseand the amylase will be produced, in at least some amounts, almostregardless of changes in the culture medium and other conditions ofgrowth of the micro-organism. The ratio of the activity of the alkalineprotease to the activity of the neutral protease in the startingmaterials and in the polymer-enzymes product is preferably about 0.25 to1.2 to one.

Another source of enzymes which can be used as starting material inaccord with the present invention is B. subtilis strain NRRL 644, B.subtilis strain NRRL 941, and B. subtilis strain 1AM 1523 (JapaneseCulture Collection). Still other B. subtilis and other micro-organismsare available which produce protease, a mixture of proteases, orprotease and amylase, at least to a limited if not optimum extent. Thesocalled Streptomyces griseus neutral protease has a broad pH activityrange and may constitute one starting enzyme for incorporation into theproducts of the invention, as may the acid protease produced byAspergillus oxyzae.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples aregiven by way of illustration only, and are not to be construed aslimited. Experimental The general procedure employed consisted ofallowing cold solutions of enzymes in appropriate buffers to reactovernight at 4 C. with cold, homogenized polymer, e.g., EMA,suspensions. EMA-21 was preferably employed, which had a molecularweight of ca. 2030,000. Other molecular weight polymers may also beused. For example, EMA l 1, having a molecular weight of about 2-3,000,and EMA 31, having a molecular weight of about 60,000, may also beemployed. Separation of soluble and insoluble adducts, after reaction,was achieved by centrifugation in the cold (Sorval SS-3 centrifuge, ca.10,000 r.p.m. and 10 min. centrifugation time). The soluble adducts weregenerally exhaustively dialyzed against water in the cold and thenlyophilized. Insoluble adducts were washed (and centrifuged), usually 10times with cold buffer and five times with cold distilled water and thenlyophilized.

The reaction of the polymer with the plurality of enzymes, as in some ofthe examples, can obviously be carried out stepwise, one enzyme at atime, with or without intermediate isolation, or with all enzymes atonce. The latter procedure is preferred for reasons of time,convenience, and economy.

EXAMPLES l-3 Soluble EMA-Trypsin (SEMAT) Trypsin (WorthingtonBiochemical Co.) was stored in the cold and was used as received. EMA-21was converted completely to the anhydride by heating at 105 C. in vacuoto constant weight (ca. 15 hr.) and then stored in sealed containersuntil used. BAEE (benzoylarginine ethyl ester) was obtained from MannLaboratories and used as received. RNa'se (bovine ribonuclease) wasobtained from Worthington Biochemical Co. and used for the reportedSEMAT digestion as received.

SEMAT In a typical experiment 500 mg. trypsin was dissolved in 15 ml. ofcold 0.2 M phosphate buffer, pH 7.5 and 1.00 g. of EMA was homogenizedin a Waring blender for ca. 1 min. with ml. cold phosphate buffer, pH7.5. The solutions were combined and the mixture was stirred in a coldroom (4 C.) for 12-15 hours. The mixture was then centrifuged in thecold for 10 min. at ca. 10,000 r.p.m. in a Sorvall SS-l centrifuge.

The supernatant was separated from the sedimented, crosslinkedTrypsin-EMA adduct (IMET) and the supernatant was exhaustively dialyzedagainst deionized water at 4 C., to tion r ine c 60 p r n of the gin i iy remove phosphate ion. Lyophilization of the dialyzed material after 2ays n luti n at r m p ratur yielded the crude product. The ratio ofsoluble to insoluble ad- Kinetic Parameters duct and the protein contentof the adduct varies with the The kinetic parameters, K and V for SEMATand trypratio of trypsin to EMA employed in the preparation (table 1 sinwith BAEE as substrate are given in table 2.

SEMAT Purification pH Activity Profiles TABLE l.SEMA'I PREPARATIONSAmount crude Amount Amount Yield, chromato- Yield Phosphate Activitytrypsin EMA-21 crude Percen graphed b product b Percent butler, pHunits/secJ (mg.) (mg.) (mg.) N- (mg.) (mg.) N- 7.5 M mg. X

1a 105 52 3. 50 0. 2 3. 95 500 1,000 291 2 83 0 02 Active 101 35 0 02Active 1 Based on dry Weight.

b Crude product chromatographed on Sephadex G-100 (crosslinked dextran;phosphate buffer, pH 7.5); product taken from fractions at void volume.I

s Activity using BAEE substrate (ca. 1.3-2.7)(10- M). Trypsin activityunder comparable conditions corresponds to 1.6)(10- units/sec./mg.Assayed at pH 9.5 (carbonate/bicarbonate).

For general purification ca. 100 mg. of crude SEMAT dis- Activity ofSEMAT samples and trypsin were made by solved in a minimum amount of 10percent sucrose/0.2 M determining the rate of hydrolysis of BAEE asmeasured by phosphate of buffer pH 7.5 and placed on a column (ca. 5X402 5 the change in absorbance at 255 mu. (table 3). cm.) of SephadexG-100 (cross-linked dextran) equilibrated with phosphate buffer, pH 7.5.Elution was followed in the TABLE usual manner and fractions werecollected. The void volume Soluble EMA/Trypsin percent Maximum BABEActivity vs eluant was collected, dialyzed and lyophilized to yieldproduct PH (Tris Buffers to PH 9; Carbonate/Bicarbonate B ff T- above pH9) The integrity of the SEMAT was shown by chromatography of physicalmixtures of trypsin and HEMA (hydrolyzed EMA) p" Twpxin SEMAT which gavea void volume fraction which had very low protein content as shown by UVabsorption at 280 mp. vs 215 my. and 6 9 83 30 low nitrogen content(less than 0.2 percent). Disk gel elec- 90 45 trophoresis toward bothpositive and negative poles did not 7- 95 65 give any migrating andstainable bands with purified SEMAT 80 whereas a mixture of trypsin andHEMA gave a band with RF :3 approximately identical with native trypsin.SEMAT gave a 9.5 90 96 stain (Amido Schwarz dye) when added to the lowergel which 40 73 97 was then polymerized and stained.

SEMAT Stability SEMAT samples were dissolved in-0.2 M phosphate bufferUnpredictably, the pH optimum of SEMAT extends into the or in 0.1 M KCland allowed to stand in solution either at g r p" rangesin contrast withnative yp room temperature or in a refrigerator (ca. 4 C.). Trypsin 7solutions were prepared in similar solutions and subjected to EXAMPLE 4identical conditions as for the SEMAT solutions. y yp EMA Activity ofthe samples (SEMAT and tr sin) was made b determining their activitiestoward hydrii l ysis of BAEE a: To 240 of cold M buffer pH measured bythe (zero order) rate of reaction. The solutions 5 added "9 of M i themixture was homogemzefd were initially adjusted to have approximatelyequal activities for 1 i Dui-mg thls. tune f Chymotrypsin or activitiesbased upon equivalent protein contents. Activity (crystaunie) wasdlssolveq m 60 cold q' The was measured as a function of time (days). inthe case of a pusqlut'ons were combmed magneucauy smiled overrifiedSEMAT preparation, 65 percent of the original (BAEE) mght m a cold Themature was then cenmfuged to activity was present after 17 days insolution at room temperaseparilie soluble Insoluble ii and the two fture whereas a trypsin solution had less than 10 percent of thelyophlhzed ovemlght. The .lyophmzed. Soluble and msolutile original(BAEE) activity after 8 days in solution at room temsystems were.exhaustively dlalyzed agalpst cold water and maperature. In anotherexample an unpurified SEMAT preparalyzed' welgm'soluble.cilymotrypsm/EMA E 1,441.82 mg. Weight-insoluble chymotrypsin/EMA(IEMAC),792.11 mg.

Chromatography of SEMAC TABLE 2-TRYPSIN gg g Mgg HYDROLY' Using theprocedure described in example 1, 100 mg. of crude SEMAC waschromatographed on a Sephadex G-lOO [PH 01Mcarbmate/bmmibwaten column,equilibrated with 0.2 M phosphate bufi'er, pH 7.5. Enzyme SABAEE) Km vmole] Elution gave essentially two peaks, one centered near the voidMxlo Mxlo MXIO Sec-X105 volume (206 ml.) and the other extending over awide volume 0.655 8.33: 1,23% 1,33 (ca. 250-353 rlnl.);1 The two pelzkeluates vere collected 11 separate y, ia yze against co water anlyophilized. ggggjgfi fiifi fj '3 g Weight fraction 1, 17.60 mg.; Weightfraction 11, 22.75 mg.

70 Assay of Chymotrypsin and SEMAC with ATEE (see infra) ;igjigg gg gfig gifg iggffi as trypsin (389% ATEE activities of chgmotrypsin andSEMfAC (fraction 1) s n unpuri e s H. Ne urath and G. W. Schwert, Chem.Rev. 40, 69 (1950). 3 2: fgg l z i g iEg zgg fl s in i g 3 :65:NOTE.-From the Michaelis-Menten equation (see item 0 above), Soacetonitme) was added to 3 m]. of phosphate buffer. At zero indicatesinitial substrate (BAEE) concentration, Km indicates the nMichaelis-Menten constant, and V'm". indicates the maximum velocity."me, micmhters f Chymotrypsin sohmon (0.398 mg TABLE 4 SolubleEMA/Chymotrypsin ATEE percent maximum activity vs pH (Tris-HCl buffersfrom pH 6.8 to 9.4; tris buffer above pH 9.4)

% Maximum Activity (Arbitrary activity units) pH Chymotrypsin SEMACUnpredictably, the pH optimum of SEMAC extends into the higher pH rangesin contrast with native chymotrypsin.

Stability Studies on Chymotrypsin, Soluble Chymotrypsin and insolubleEMA-Chymotrypsin Chymotrypsin (2.00 mg.), soluble EMA-chymotrypsin(60.02 mg.) and insoluble EMA-chymotrypsin (30.08 mg.) were eachdissolved in 2 ml. of 0.2 M phosphate buffer, pH 7.5, and allowed tostand at room temperature. Esterase activities were measured over aperiod of days. Esterase activities were measured by determining therate of hydrolysis of N- acetyl-L-tyrosine ethyl ester (ATEE) asdetermined by following the change in absorbance at 237 mp. of asolution as a function of time. For assay, 100 microliters of a solutionof ATEE (40.06 mg. in 2 ml. acetonitrile) was added to 3 ml. ofphosphate buffer and hydrolysis was initiated by addition of 10microliters of the chymotrypsin or soluble EMA- chymotrypsin solutionsand by addition of 25 gamma of the insoluble EMA-chmotrypsin. By thismethod, after 2 days at room temperature, chymotrypsin had 60 percent ofits initial activity, the insoluble EMA-chymotrypsin had 97 percent ofits initial activity, and the soluble EMA-chymotrypsin had 99 percent ofits initial activity. After 7 days at room tempera ture, the solubleEMA-chymotrypsin solution had 93 percent of its initial esteraseactivity.

EMA-

EXAMPLE Lipase-EMA and Amylase-EMA Lipase (MY-20, TM-Meito-Sangyo Co.Ltd., Tokyo, Japan) (251.32 mg., 25 ml. 0.2 M phosphate bufier) wasadded to homogenized EMA-21 (l min. homogenization time) in thephosphate buffer (500.61 mg., 50 ml. buffer) and allowed to reactovernight. After dialysis, lyophilization and isolation there wasobtained 506.87 mg. soluble adduct and 1 l 1.18 mg. insoluble adduct.

Lipase Assay Procedure The substrate for the assay was olive oil. Anemulsion was prepared as follows: Polyvinyl alcohol (20 g.) (98 percenthydrolyzed; Matheson, Coleman and Bell) was stirred in 1 liter distilledwater at 75 C. until dissolved, then cooled and neutralized to pH 7.0with NaOH. Olive oil (10 ml.) and 90 ml. polyvinyl alcohol solution werehomogenized in a Waring Blendor for 5 minutes and then the pH wasadjusted to 7.0 with N/ l NaOH. A fresh suspension was prepared everyday. The enzyme assay was carried out using the Radiometer pH- stat.This equipment comprised a No. 25 pH meter, a No. 1 l titrator, a ABUlbauto burette and a SBR2c titrigraph. The volume of the burette was 0.25ml. Olive oil emulsion (3 ml.) was placed in the titration vessel atroom temperature. The reaction was generally initiated by addition of0.25 ml. of lipase or lipase/EMA solution (2-4 mg./ml.) and the rate ofbase uptake (0.01 N NaOH) required to maintain the pH at a constantvalue was measured. A stream of CO -lrce nitrogen gas was used to flushCO from the reaction chamber and a magnetic stirrer was used to ensureadequate mixing of the system. The rate of hydrolysis was measured fromthe linear portion of the titration graph and expressed as 2 moles fattyacid liberated per minute per mg. enzyme.

Weight based upon protein with lipase; total weight sample forlipase/EMA systems.

Assay values ranged from 2-4 moleslminJn-lg. for several samples. Thevalues were reproducible, however, within a given sample. No explanationcan be given for this anomaly.

An advantageous purification of the crude enzyme which frequently occursduring covalent bonding of an enzyme to a polymer molecule accounts forthe high activity of the insolubles. pH Profile of Lipase and InsolubleLipase/EMA Lipase activities were determined for lipase and insolublelipase/EMA using the olive oil emulsion. The pH of the emulsion wasadjusted immediately prior to use by addition of 0.1 or 0.01 M sodiumhydroxide. The same emulsion, at appropriate pH, was used for both thelipase and lipase/EMA. A correction for spontaneous base-catalyzedhydrolysis was made at the higher pH values by taking initial slopes andsubtracting this value from the zeroorder enzyme catalyzed reac tion.Results are tabulated in table 5.

in exactly the manner of the foregoing example, amylase- EMA is preparedusing bacterial amylase from B. subtilis AM and EMA-21. ln replicateexperiments, the amylolytic activity of the soluble and insolubleEMA-amylase products varies between 44 and 72 percent of that of theparent native enzyme.

TABLE 5 Lipase Activity vs pH (activity in mmoles/min./mg. X 10)Cellulase-EMA Cellulase (Cellosin AP, TM-Nomoto Co., Japan) (4.001 g.added to 60 ml. cold water, with incomplete dissolution) was added to asuspension of EMA-21 (1.001 g. with 240 ml. cold 0.2 M phosphate buffer,pH 7.48 homogenized for 1 minute) and the mixture stirred overnight at 4C. The mixture was centrifuged and the supernatant and insolublematerials were dialyzed exhaustively against water, and lyophilized.Weightsoluble cellulase/EMA, 0.9997 g.; weight-insoluble cellulase- EMA,1.8786 g. Cellulase Assay Procedure The assay procedure was essentiallythat of Worthington Biochemical Corp. Freehold, NJ. and determines theamount of glucose liberated per unit time as measured by the Glucostat(reagent kit for glucose assay) system (Worthington Biochemical Corp.).Enzyme solution a weighted amount of cellulase or cellulase/EMA wasadded to 0.05 M sodium acetate and stirred and diluted as required.

Substrate solution A ca. 1.2 percent carboxymethylcellulose solution(1.200 g., Cellulose Gum, Type 70 Premium, medium, Hercules Powder Co.)was prepared by dissolution in 80 ml. hot 0.05 M citrate buffer, pH 3.5.After dissolution the solution was cooled and the volume adjusted to 100ml. The final pH was ca. 4.

Standard solution An aqueous solution was prepared such that theconcentration of D-glucose was ca. 0.05-0.3 mg./ml.

Glucostat solution The contents of the Chromagen (color developer) vialwere dissolved in distilled water and the volume adjusted to 60 ml. Thecontent of the Glucostat vial was dissolved in the solution and thevolume adjusted to 90 ml.

Assay Test solutions were made by taking 1 ml. of the enzyme solutionand adding to ml. of substrate. From each solution 1 ml. was taken.Standard solutions were 1 ml. aliquots of the glucose solutions. To thereaction mixtures was added 9 ml. of the Glucostat reagent. The reactionwas allowed to proceed for 1 hr. at room temperature and the reactionwas then stopped by addition of one drop of 4 N HCl. After 10 minutesthe optical density of the solutions were read at 400 mp. using areagent blank.

Activity g. glucose/hL/mg. engymc system Cellulosin'AP (commercialcellulose) 0.048 Insoluble cellulite/EMA 0.014 Soluble cellulase/EMA0.l57

This result is due to a purification of the crude enzyme by attachmentto the polymer,'which accounts for the high activity of the solubles.

EXAMPLE 7 B. subtilis Neutral and Alkaline Protease-EMA insoluble andSoluble Adducts B. subtilis alkaline and neutral proteases (200 mg.) aredissolved in 50 ml. cold 0.] M in phosphate and 0.01 M in calciumacetate, pH 7.5, and this solution is then added to a cold, homogenizedmixture of EMA-21 (100 mg.) suspended in 50 ml. 0.! M phosphate pH 7.5.The mixture is stirred overnight in the cold (4 C.) and the insolublematerial is separated from the supernatant by centrifugation. Afterwashing the solids.

five times with cold 0.] M NaCl and twice with water, the material islyophilized to yield a solid which possesses 38 percent of neutralprotease activity and 52 percent of the original alkaline proteaseactivity.

The supernatant solution is dialyzed against cold, distilled water andthen lyophilized to yield a soluble solid which possesses 47 percent ofthe neutral protease activity and 59 percent of the original alkalineprotease activity.

The ratio of the alkaline protease activity to the neutral proteaseactivity in the starting material and in the polymerenzyme product ispreferably about 0.25-l .2 to 1.

EXAMPLE 8 B. subtilis Neutral and Alkaline Protease and Lipase-EMAinsoluble and Soluble Adducts.

B. subtilis neutral and alkaline proteases (400 mg.) and Lipase (400mg., Lipase-MY Meito-Sangyo Co., Japan) are dissolved in 300 m. cold 0.2M in phosphate and 0.01 M in calcium acetate, ph 7.5, and this solutionis added to a cold, homogenized mixture of EMA-2i (200 mg.) suspended inml. of 0.1 M phosphate, pH 7.5. The mixture is stirred overnight in thecold (4 C.) and the supernatant is separated from the insoluble materialby centrifugation. After washing the solid with 0.1 M NaCl and waterfollowed by lyophilization there is obtained a solid which possesses 35percent of the neutral protease activity, 47 percent of the alkalineprotease activity, and 72 percent of the original lipase activity.

Dialysis of the supernatant against cold, distilled water followed bylyophilization yields a soluble solid which possesses 43 percent of theneutral protease activity, 56 percent of the alkaline protease activity,and 17 percent of the original lipase activity.

EXAMPLE 9 Oxynitrilase-EMA Oxynitrilase was isolated from bitter almonds(Tri-Co Almond Co., Chico, Calif.) according to the procedure of Pfeiland Becker, J. Am. Chem. Soc. 88, 4299 (1966). The crude enzyme obtainedby ethanol precipitation was rechromatographed on Sephadex (cross-linkeddextran) prior to use. In ml. cold 0.2 M phosphate buffer pH 7.5, wasdissolved 307.60 mg. oxynitrilase. EMA-2l (613.12 mg.) was homogenizedin a blender for 1 min. with 60 ml. cold phosphate buffer. To the EMA-2ihomogenate was added 10 ml. of 1 percent hexamethylene diamine in waterand the mixture was stirred for 2 min., after which time theoxynitrilase solution was added and the mixture was stirred overnight at4 C. The mixture was then centrifuged to separate supernatant from thesolids. The solids were washed five times with cold phosphate buffer andthen eight times with cold water. The solids and the supernatant werethen dialyzed and lyophilized. Weight soluble Oxynitrilase-EMA: 428 mg.;weight-insoluble oxynitrilase-EMA: 838.07 mg.

Anal: Insoluble oxynitrilase-EMA: %N, 3.60; 3.68.

Assay of Insoluble Oxynitrilase-EMA Activities were determined byfollowing optical rotation as a function of time using a Perkin-Elmerinodel l4l Polarimeter, with a l decimeter cell; at the D-line ofsodium.

1. Crotonaldehyde/Sodium Cyanide: To 5 ml. of a solution of sodiumcyanide (7.6 g. in 250 ml. distilled water) adjusted to pH 5.4 with 6 Nacetic acid, was added 1 ml. ofa water slurry of oxynitrilase-EMA (10mg. per 2 ml.). At zero time 5 ml. of a crotonaldehyde solution (7 g. in250 ml. distilled water) was added and the mixture stirred for a fewminutes prior to recording of optical rotation.

After 5 minutes the optical rotation was a],,-0.048 which indicates theOxynitrilase-EMA is catalyzing the formation of the optically activecyanhydrin of crotonaldehyde.

2. DL-Mandelonitrile: in 200 ml. 50 percent methanol/0.05 M sodiumacetate, adjusted to an apparent pH 5.3 with 6 N acetic acid, wasdissolved l 1.0 g. DL-mandelonitrile. The system was maintained under anitrogen purge to prevent formation of benzoic acid from benzaldehyde.At zero time 21 [.07 mg. of insoluble oxynitrilase-EMA was added andafter brief stirring an aliquot was removed and optical rotation wasrecorded.

After 2 minutes the optical rotation was ad -0.012; after 3 minutes itwas al,,-0.045; after 5 minutes a],,-0.066, which indicatesthe-oxynitrilase-EMA is catalytically converting D- mandelonitrile tobenzaldehyde plus hydrogen cyanide while the L-mandelonitrile remainsessentially unchanged.

3. Crotonaldehyde/Hydrogen Cyanide: Freshly distilled hydrogen cyanide(15 ml.) was dissolved in 500 ml. of cold water. Crotonaldehyde (1.408g.) was dissolved in 100 ml. of cold water (0.2 M). To 5 ml. of thecrotonaldehyde solution was added 1 1.20 mg. of Oxynitrilase-EMA. Atzero time 5 ml. of the hydrogen cyanide solution was added, the mixturewas stirred briefly and the optical rotation was then recorded.

After 2 minutes the optical rotation was oil -0.006; after 4 minutes, ad-0.059; after 10 minutes nth-0.204, which indicates the oxynitrilase-EMAis catalyzing the formation of the optically active cyanhydrin ofcrotonaldehyde.

4. Glycolaldehyde/Hydrogen Cyanide: Glycolaldehyde (1.206 g., K and KLaboratories) was dissolved in 100 ml. water (0.2 M). To 5 ml. of thissolution was added 10.47 mg. oxynitrilase-EMA. At zero time, 5 ml. ofthe above hydrogen cyanide solution was added and, after brief stirring,the optical rotation was recorded.

After 2 minutes the optical rotation was fl] 0.0O9; after 4 minutes, ad-0.055; after 7 minutes ad -0.117, which indicates the oxynitrilase-EMAis catalyzing the formation of the optically active cyanhydrin ofglycolaldehyde.

Assay of Soluble EMA-Oxynitrilase ln 2 ml. distilled water was dissolved10 mg. soluble EMA- oxynitrilase. in 125 ml. water was dissolved 3.5 g.crotonaldehyde. in 125 ml. cold water was dissolved 3.8 g. sodiumcyanide and the pH was adjusted to 5.4 with 6 N acetic acid. To 5 ml. ofthe cyanide solution was added 1 ml. of the soluble EMA-oxynitrilasesolution. At zero time 5 ml. of the crotonaldehyde solution was added tothe mixture and the rotation (in negative millidegrees) was followed asa function of time (min.) in a Perkin-Elmer model 141 polarimeter usinga one decimeter quartz cell. At 2 min.a] 0.272; 5 min. (fl -0.317; 10min. ad -0.371, showing that the soluble EMA-oxynitrilase productpossesses the enzymatic activity of the native enzyme in conversion ofHCN and crotonaldehyde to an optically active crotonaldehydecyanohydrin.

EXAMPLE 1o Asparaginase-EMA Asparaginase (Worthington, 20 units/mg,minimum) (13.55 mg.) was dissolved in 10 ml. cold 0.2 M phosphatebuffer, pH 7.5. EMA-21 (50.28 mg.) was homogenized for 45 sec. with 50ml. phosphate bufi'er, added (with an additional ml. cold buffer) to 0.5ml. 1 percent hexamethylenediamine and stirred for 45 sec., at whichtime the cold asparaginase solution was added and the mixture wasstirred overnight at 4 C. Workup in the usual manner of centrifugation,dialysis and lyophilization gave the soluble and insoluble systems.Weight of soluble asparaginase-EMA: 25.29 mg.; weight of insolubleasparaginase-EMA: 57.55 mg.

Asparaginase Assay Procedure The procedure of Worthington BiochemicalCorp. was employed. Enzyme and EMA soluble and insoluble concentrationswere 1 mg./l ml. physiological saline. L-Asparagine substrate was 0.01 Min 0.05 M tris-HCl [tris(hydroxymethylaminomethane)hydrochloride]buffer, pH 8.6. A solution consisting of 1.7 ml. substrate and 0.2 ml.tris-HCl buffer were incubated at 37 C. At zero time, 0.1 ml. enzyme orenzyme/EMA solution was added and the mixture was incubated at 37 C. for10 min. At the end of this time 0.1 ml. 1.5 M trichloroacetic acid wasadded, the mixture centrifuged, and 0.5 ml. of the supernatant was addedto 7.0 ml. water followed by 1.0 ml. Nessler's reagent. After 10 min.absorbance was read at 480 mp. A control was a sample which thetrichloroacetic acid was added to the substrate prior to enzymeaddition. Standards were ammonium sulfate solutions that contained 0.5to 0.1 p.mole nitrogen per 0.5 ml.

Assay pH 8.6 I

Asparaginase-71 units/ mg. protein. Insoluble Asparaginase-EMA 1.5units/mg. total weight. Soluble Asparaginase--EMA 27 units/mg. totalweight.

Anal: soluble asparaginase/EMA contained 5.48% N, dry weight.

EXAMPLES 11 and 12 Pepsin-EMA A. 1:1 weight ratio, pepsimEMA: In 150cold 0.2 M acetate buffer, pH 5.0, was dissolved 201.03 mg. pepsin(Sigma Chemicals; 1160,000). This solution was added to a mixture of203.41 mg. EMA2l in 50 ml. cold buffer which had been homogenized for 1min. The combined solutions were stirred overnight at 4 C. Thesupernatant and solids were separated by centrifugation, the supernatantdialyzed against cold, distilled water and the solid material washedtwice with cold water and then dialyzed overnight. The supernatant andsolid fractions were then lyophilized to give 129.35 mg. solublepepsin-EMA (7.25% N, dry weight) and 32.20 mg. insoluble pepsin-EMA(1.16% N, dry weight). (Washing the solids with buffer sodium chloridesolutions resulted in what appeared to be disperse gels.)

B. 4:1 weight ratio, pepsin: EMA: Using the same procedure as above,403.38 mg. pepsin in l00 ml. cold 0.05 M acetate buffer was allowed toreact with 101.21 mg. EMA homogenized with 125 ml. cold acetate buffer.The solid isolated after contrifugation was washed twice with waterfollowed by dialysis, as was the supernatant. Lyophilization of the twofractions gave 226.39 mg. soluble pepsin-EMA (12.2% N, dry weight) and68.11 mg. of insoluble pepsin- EMA (0.32% N, dry weight).

Assay Procedure Hemoglobin (2.5 g.) was added to ml. distilled water andhomogenized for 30 sec. and then filtered through glass wool. The pH wasadjusted by addition of the appropriate buffer. Assay of the pepsin andpepsin-EMA systems then proceeded according to the procedure ofWorthington Biochemical Corp. whereby the hydrolysis of hemoglobin ismeasured per unit time at 37 C. as determined by the optical density at280 mp. of the trichloroacetic acid soluble materials. Activities areexpressed in units/mg. weight pepsin or pepsin-EMA. (One unitcorresponds to a 280 my. absorbancy of TCA-soluble hydrolysis productsof 0.001 per minute at 37).

pH Profile-Activity Assays were based upon hemoglobin activities as afunction of pH. Buffers employed were HCl-KCl from pH 1.0-3.1]

(l=0. l 0) and sodium acetate from pH 3.60-5.8 (l=0.05).

TABLE 6 Pepsin-EMA Derivatives/pH Profiles Maximum Activity (HemoglobinAssay) Soluble EMA-peplin lnlol. EMA-Pepsin P Pepsin (7.25% N) (0.35% N)too 100 92 Buffer! employed: HCl-KC] (l=0.l0) from pH 1.0-3.1;sodiu1r1acetate (l=0.05) from pH 3.6-5.8.

EXAMPLE 13 Water-soluble EMA-Papain, Mercuri and Zinc Papain A. SolubleEMA-Mercuri and Zinc Papain In 50 ml. of cold 0.0l M phosphate buffer,pH 7.0, containing 0.002 M in cysteine, is dissolved 250 mg. commercialHgpapain. To this solution is added a homogenized mixture of EMA-21 (200mg. EMA in 100 ml. cold 0.01 M phosphate, pH 7.0, containing 0.002 Mcysteine). The mixture is stirred overnight in the cold (4 C.) and thencentrifuged for min. (8,000 r.p.m. to separate insoluble materials. Thesupernatant solution is then exhaustively dialyzed against cold waterwhich is 0.002 M in cysteine. Lyophilization of the dialyzed materialgives an aqueous soluble product which possesses65 percent of theprotease activity (after the mercury is removed in the Anson variationof the Kunitz casein test method J. Gen. Physiol. 30, 291 (1947),employing EDTA (ethylene diamine tetraacetic acid) in the presence ofcrysteine).

1n the same manner, the water-soluble EMA-zinc papain is prepared fromEMA- and zinc-papain (US. Pat. No. 3,284,316) and found to have 40percent of the initial protease activity after removal of the zinc; 10percent before removal of zinc.

B. Soluble EMA-Papain In 25 ml. cold 0.01 M phosphate buffer, pH 7.0,which is 0.002 M in cysteine, is dissolved 100 mg. of crude papain. Tothis cold solution is added an homogenized mixture of 100 mg. EMA-21 in100 ml. cold 0.01 M phosphate, pH 7.0 which is 0.002 M in cysteine. Themixture is stirred overnight in the cold (4 C.), centrifuged, and thesupernatant is exhaustively dialyzed against cold 0.002 M cysteine.Lyophilization of the dialyzed material gives an aqueously solubleEMA-papain product which possesses percent of the protease activity.

EXAMPLE l4 Lipase-Styrene Maleic Anhydride Copolymers Coupling ofbacterial lipase to an alternating styrene-maleic anhydride (1:1)copolymer, in aqueous buffer medium using the conventional procedure ofexamples 1 to 12 at carrier to enzyme ratios of 1:15 to 3:1, yieldssoluble polymer-lipase derivatives having up to about percent of theoriginal enzymatic activity.

EXAMPLE l5 Cellulase/Protease-Vinyl Methyl Ether/Maleic AnhydrideCopolymers Coupling of bacterial cellulase plus B. subtilis AM neutraland alkaline bacterial proteases to an alternating vinyl methylether-maleic anhydride (1:1) copolymer, in aqueous bufier medium usingthe conventional procedure of examples 1 to 12 at carrier to enzymeratios of 1:15 to 3:1, yields soluble polymer-cellulase/neutralprotease/alkaline protease derivatives having up to about 50 percent ofeach of the original enzymic activities.

EXAMPLE 16 cellulase-Vinyl Acetate/Maleic Anhydride Copolymers Couplingof bacterial cellulase to an alternating vinyl acetate-maleic anhydride(1:1) copolymer, in aqueous buffer medium using the conventionalprocedure of examples 1 to 12 at carrier to enzyme ratios of 1:15 to3:1, yields soluble polymer-cellulase derivatives having up to about 60percent of each of the original enzymic activities.

EXAMPLE 17 Cellulase/Lipase/Protease-Divinyl Ether/Maleic AnhydrideCyclocopolymers Coupling of bacterial cellulase, lipase, and alkalineprotease to a divinyl ether-maleic anhydride cyclocopolymer (havingrepeating units consisting of adjacent ethylene-maleic anhydridesegments which are additionally bonded to each other by ether linkage),in aqueous bufi'er medium using the conventional procedure of examples 1to 12 at carrier to enzyme ratios of 1:15 to 3:1, yields solublepolymer-cellulase/lipase/protease derivatives having up to about 50percent of each of the original enzymic activities.

EXAMPLE 18 Chymotrypsin-Polymaleic Anhydride Polymers Coupling ofchymotrypsin to a polymaleic anhydride polymer, in aqueous buffer mediumusing the conventional procedure of examples 1 to 12 at carrier toenzyme ratios of 1:15 to 3:1, yields soluble polymer-chymotrypsinderivatives having up to about 70 percent of the original enzymicactivity.

EXAMPLE l9 Trypsin-Polymaleic- Anhydride Polymers Coupling of trypsin toa polymaleic anhydride polymer, in aqueous buffer medium using theconventional procedure of examples 1 to 12 at carrier to enzyme ratiosof 1:15 to 3:1. yields soluble polymer-trypsin derivatives having up toabout 70 percent of the original enzymic activity.

EXAMPLE 20 Protease-Polymaleic Anhydride Polymers Coupling of a mixtureof alkaline and neutral proteases produced by B. subtilis to apolymaleic anhydride polymer, in aqueous buffer medium using theconventional procedure of examples 1 to 12 at carrier to enzyme ratiosof 1:15 to 3:1, yields soluble polymer-alkaline protease/neutralprotease derivatives having up to about 70 percent of the originalenzymic activities.

EXAMPLE 21 Acid Protease-Polyacrylic Anhydride and EMA Polymers Couplingof acid protease produced by Aspergillus oryzae to a polyacrylicanhydride polymer, in aqueous buffer medium using the conventionalprocedure of examples 1 to 12 at carrier to enzyme ratios of 1:15 to3:1, yields soluble polymerprotease derivatives having up to about 50percent of the original enzymic activity.

In the same manner, the identical enzyme-polymer product is producedfrom polyacrylic acid, using Woodward's reagent, N-ethyl-S-phenylisooxazolium-3-sulfonate, as activator for the carboxyl groups of thepolyacrylic acid.

Moreover, direct reaction of the acid protease from A. aryzae withEMA-21 in the manner of examples 1 to 12 produces soluble EMA-acidprotease having an exceptional activity at acid pH's, the percentage ofacid proteolytic activity based upon starting native acid proteasevarying between about 23 and 68 percent.

EXAMPLE 22 B. subtilis Neutral and Alkaline Proteases and Amylase/EMAInsoluble and Soluble Adducts.

B. subtilis neutral and alkaline proteases and amylase mixture (250 mg.)is dissolved in 100 ml. cold 0.1 M in phosphate and 0.01 M in calciumacetate, pH'7.5, and to this solution is added a homogenized mixture ofEMA-21 (200 mg.) suspended in 50 ml. cold 0.1 M phosphate, pH 7.5. Themixture is stirred overnight in the cold (4 C.) and the insolublematerial is separated from the supernatant by centrifugation. Afterwashing the solids five times with cold 0.1 M NaCl and twice with water,the material is lyophilized to yield a solid which possesses 32 percentof the original neutral protease activity, 48 percent of the originalalkaline protease activity, and' 62 percent of the original amylaseactivity.

The supernatant solution is dialyzed against cold, distilled water andthen lyophilized to yield a soluble solid which possesses 42 percent ofthe original neutral protease activity, 57 percent of the originalalkaline protease activity, and 69 percent of the original amylaseactivity.

The ratio of the activity of the alkaline protease to the activity ofthe neutral protease in the starting materials and in the polymer-enzymeproducts is preferably about 0.25 to 1.2 to 1.

EXAMPLE 23 Bacillus subtilis Neutral Protease and Dextranase-EMA Solubleand Insoluble Products B. subtilis neutral protease (100 mg.) anddextranase (100 mg.) are dissolved in 75 ml. cold 0.1 M phosphatebuffer, pH

7.5, which is also 0.0l M in calcium acetate. To this solution is addeda homogenized mixture of EMA-21(250 mg.) in 100 ml. cold 0.1 M phosphatebuffer, pH 7.5. The combined mixture is stirred overnight in the cold (4C.) and the solid is separated from the supernatant solution bycentrifugation. After dialysis and lyophilization the insoluble B.subtilis neutral protease and dextranase-EMA product possesses 43percent of the original protease activity and 27 percent of the originaldextranase activity.

The supernatant solution is dialyzed and lyophilized to yield a solublesolid B. subtilis neutral protease and dextranase- EMA product whichpossesses 64 percent of the original protease activity and 62 percent ofthe original dextranase activity. 1

EXAMPLE 24 Dextranase-EMA Soluble and Insoluble Products.

Dextranase was obtained as a solid precipitated by organic solventaddition to a fermentation beer of Penicillium funiculosum (strain NRRL1132).

Dextranase (100 mg.) is dissolved in 50 ml. cold 0.1 M phosphatebufi'er, pH 7.5. which is 0.0] M in calcium acetate. To this solution isadded a homogenized mixture of EMA-21 (I mg.) in 50 ml. cold 0.1 Mphosphate buffer. The combined mixture is stirred overnight in the cold(4 C.) and the solid is separated from the supernatant solution bycentrifugation. After dialysis and lyophilization the insolubledextranase- EMA product possesses 35 percent of the original dextranaseactivity.

The supernatant solution is dialyzed and lyophilized to yield a solublesolid dextranase-EMA product which possesses 68 percent of the originaldextranase activity.

EXAMPLE 25 B. subtilis Neutral and Alkaline Proteases and Amylase/D-MAPAl-EMA The partial dimethylaminopropylamine imide of EMA-21 wasprepared by refluxing a mixture of EMA and a limiting amount (50 percentby weight) of N,N- dimethylaminopropylamine in xylene for 5 hours.During this time water was removed using a Dean-StarK trap. After waterevolution had ceased, indicating completion of the reaction, the productwas isolated by precipitation with hexane and drying in vacuo at 105 C.The imide product contained 8.9% N, indicating an imide content of 54percent.

The B. subtilis AM neutral and alkaline protease and amylase mixture(500 mg.) is dissolved in 50 ml. cold 0.] M phosphate bufi'er, pH 7.5,which is also 0.02 M in calcium acetate. To this solution is added acold mixture of the DMAPAl-EMA (500 mg.) which is homogenized for lminute with 50 ml. cold 0.] M phosphate buffer, pH 7.5. The combinedmixture is stirred overnight in the cold (4 C.). The insoluble productis separated from the supernatant by centrifugation and then washedeight times with 0.1 M NaCl and five times with water and thenlyophilized. The insoluble enzymepolymer product thus obtained possesses36 percent of the original neutral protease activity, 42 percent of theoriginal alkaline protease activity, and 52 percent of the originalamylase activity.

The supernatant solution is dialyzed against water and lyophilized toyield the soluble enzyme-polymer product which possesses 46 percent ofthe original neutral protease activity, 48 percent of the originalalkaline protease activity, and 50 percent of the original amylaseactivity.

The ratio of the activity of the alkaline protease to the activity ofthe neutral protease in the starting materials and in the cationicpolymer-enzyme product is preferably no greater than about0.25 to 1.2 to1.

EXAMPLE 26 Trypsin/DMAPAl-EMA The partial dimethylaminopropylamine imideof EMA-2] was prepared by refluxing a mixture of EMA and a limitingamount percent by weight) of N ,N- dimethylaminopropylamine in xylenefor 5 hours. During this time water was removed using a Dean-Stark trap.After water evolution had ceased, indicating completion of the reaction,the product, was isolated by precipitation with hexane and drying invacuo at l05 C. The imide product contained 8.9% N, indicating an imidecontent of 54 percent.

Trypsin (250 mg.) is dissolved in I00 ml. cold 0.l M phosphate buffer,pH 7.5, and to this solution is added a mixture of the DMAPAl-EMA (250mg.) which is homogenized for 1 minute with I00 ml. cold 0.] M phosphatebuffer, pH 7.5. The combined mixture is stirred overnight in the cold (4C.) and the insoluble polymer-enzyme product is separated from thesupernatant solution by centrifugation. After washing eight times with0.l M NaCl and five times with water, the soluble polymer-enzyme productis obtained by lyophilization and possesses 55 percent of the originaltrypsin activity as measured at pH 7.5.

The supernatant solution is dialyzed against water and lyophilized toyield a soluble polymer-enzyme product which possesses 65 percent of theoriginal trypsin activity as measured at pH 7.5.

The soluble trypsin-polymer product and insoluble trypsinpolymer productboth possess greater enzymatic activities at lower pH ranges as comparedwith the native enzyme.

EXAMPLE 27 Asparaginase/DMAPAl-EMA The partial dimethylaminopropylamineimide of EMA-21 was prepared by refluxing a mixture of EMA and alimiting amount (50 percent by weight) of N,N- dimethylaminopropylamineih xylene for 5 hours. During this time water was removed using aDean-Stark trap. After water evolution had ceased, indicating completionof the reaction, the product was isolated by precipitation with hexaneand drying in vacuo at 105 C. The imide'product contained 8.9% N,indicating an imide content of 54 percent.

Asparaginase (34 mg.) is dissolved in 30 ml. cold 0.] M phosphatebuffer, pH 7.5 and to this solution is added a mixture of the DMAPAl-EMA(45 mg.) which is homogenized for 1 minute with 30 ml. cold 0.1 Mphosphate buffer, pH 7.5. The combined mixture is stirred overnight inthe cold (4 C.) and the insoluble polymer-enzyme product is separatedfrom the supernatant solution by centrifugation. After washing eighttimes with 0.1 M NaCl and five times with water the insolublepolymer-enzyme product is obtained by lyophilization and possesses l 1percent of the original asparaginase activity.

The supernatant solution is dialyzed against water and lyophilized toyield a soluble polymer-enzyme product which possesses 36 percent of theoriginal asparaginase activity.

Other N,N-dilower-alkylaminolower-alkylamines are employed to producecorresponding additional trypsin or neutral and alkaline protease andamylase or asparaginase/dilower-alkylaminolower-alkylimide/EMA productsas the case may be, depending on the enzyme or enzyme mixture employed.

Further, for preparation of water-soluble cationic polymerenzymeproducts, polymers having such groups present in the molecule areemployed, as already illustrated.

Partial imides of a starting carboxyl or carboxylic acid anhydridecontaining polymer, e.g., EMA, are produced by:

A. Heating a limiting amount of a secondary or tertiaryaminolower-alkylamine with a water solution of the hydrolyzed orcarboxyl-containing form of the polymer in vacuo at a temperature ofabout l40-150 C. until a constant weight has been reached and water isno longer given off. Such a reaction simultaneously results in formationof imide groups and reformation of the anhydride groups. In this mannerimide-polymer products are formed which possess 5-95 percent imidelinkages, the remaining carboxyl groups (i.e., -5 percent, respectively)being present in the polymer as anhydride groups, the exact proportionsbeing dependent upon the relative amounts of starting amine and polymer.

B. Alternatively, a partial amide-polymer product may be converted tothe partial imide-polymer product by heating a partial amide-polymerproduct in vacuo at l40l50 C. until water is no longer given off. Suchan imide-polymer product likewise possesses comparable proportions ofimide and anhydride groups depending upon the number of amide groupsoriginally contained in the starting partial amide-polymer product.

Partial secondary and tertiary aminolower-alkylamides of the startingcarboxyl or carboxylic acid anhydride containing polymer, e.g., EMA, areobtained by contacting the polymer with a limiting amount of theselected amine in suspension in a solvent such as benzene or hexane,resulting in formation of a partial amide-anhydride derivative of thepolymer, or a corresponding amide-carboxylate product thereof. Thenumber of amide groups is dependent upon the quantity of the amine usedas compared with the quantity of polymer employed. Such amide-polymerproducts possess -95 percent amide groups, with remaining carboxylgroups being present as anhydride groups.

Partial aminoester-polymer products are most conveniently prepared byheating at reflux temperatures overnight a limiting quantity of theselected aminoalcohol and carboxyl or carboxylic acid anhydridecontaining polymer, e.g., EMA, in a dry organic solvent such as tolueneor dimethylformamide. The resulting product contains ester groups,carboxylic acid groups and anhydride groups, the respective numbers ofwhich are determined by the quantity of aminoalcohol used in relation tothe amount of polymer employed. Suitable blocking and unblocking of theamine moiety may be effected when required.

These products are reacted with the selected enzyme according to theprocedure of the foregoing examples to give the desired activewater-soluble cationic polymer-enzyme product.

For nonionic polymer-enzyme products, neutral groups may be attached tothe polymer molecule after enzyme attachment, e.g., alkylamines,aminoalcohols, and alcohols may be attached via reaction with residualcarboxylic or carboxylic acid anhydride groups of the polymer in theusual fashion.

Thus, in the foregoing manner, the following additional water-solubleproducts are prepared, the polymer in each case having cationicsubstituents: enzymedilower-alkylaminolower-alkanol esters of any of thepolymers employed in the foregoing examples,enzyme-lower-alkylaminolower-alkanol esters of any of the polymersemployed in the foregoing examples, and enzymeaminoloweralkanol estersof any of the polymers employed in the foregoing examples; e.g. thealkaline protease-dimethylamino propanol ester of EMA, the neutralprotease-ethylaminobw tanol ester of EMA, and the papain-aminoethanolester of polymaleicor polyacrylic anhydride or acid;enzymedilower-alkylaminolower-alkylimides of any of the polymersemployed in the foregoing examples,enzymelower-alkylaminolower-alkylimides of any of the polymers employedin the foregoing examples, and enzyme--aminolower-alkylimides of any ofthe polymers employed in the foregoing examples, e.g., the alkalineprotease-diethylamino propylimide of EMA, the neutralprotease-methylaminobutylimide of EMA, and the papain-aminopentylimideof polymaleic or polyacrylic anhydride or acid;enzymedilower-alkylaminolower-alkylamides of any of the polymersemployed in the foregoing examples,enzyme-lower-alkylaminolower-alkylamides of any of the polymers employedin the foregoing examples, enzymeaminolower-alkylamides of any of thepolymers employed in the foregoing examples, e.g., the alkalineproteasedimethylaminopropylamide of EMA, the neutralproteaseethylaminohexylamide of EMA, and the papainaminopropylamide ofpolymaleic or polyacrylic anhydride or acid.

It is apparent from the foregoing that the preferred polymer-enzymeproducts of the invention are those watersoluble products wherein thepolymer is selected from the group consisting of A. ethylene/maleicanhydride copolymer, styrene/maleic anhydride copolymer, vinyl methylether/maleic anhydride copolymer, vinylacetate/maleic anhydridecopolymer, divinyl ether/maleic anhydride cyclocopolymer, polymaleicanhydride, polyacrylic anhydride, and cationic derivatives thereof, andwherein the enzyme moiety comprises at least one enzyme selected fromthe group consisting of B. neutral protease, acid protease, alkalineprotease, lipase, cellulase, dextranase, amylase, and asparaginase, andpreferably wherein the enzyme moiety or moieties are entirely ofmicrobiological origin.

Where a protease is named specifically in the foregoing, the specificprotease may obviously be present along or in addition to anotherprotease, whether specifically named or encompassed generically by theterms acid protease. neutral protease, and alkaline protease, as well asin addition to one or more nonproteolytic enzymes.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds compositions, orprocedures shown and described, as obvious modifications and equivalentswill be apparent to one skilled in the art, and the invention istherefore to be limited only by the full scope of the appended claims.including the application of the doctrine of equivalents thereto.

We claim:

1. A water-soluble enzymatically active polymer-enzyme product whereinthe enzyme is bound covalently through groups which are nonessential forenzymatic activity to (a) a water-soluble polymer comprising chains ofcarboxylic acid or carboxylic acid anhydride units, or (b) awater-soluble polymer comprising units of carboxylic acid or carboxylicacid anhydride groups separated by carbon chains of at least one and notmore than four carbon atoms, said carbon chains being part of a unitwhich contains a maximum of 18 carbon atoms, wherein the polymer isselected from the group consisting of ethylene/maleic anhydridecopolymer, styrene/maleic anhydride copolymer, vinyl methyl ether/maleicanhydride copolymer, vinyl acetate/maleic anhydride copolymer, divinylether/maleic anhydride cyclocopolymer, polymaleic anhydride, polyacrylicanhydride, and cationic derivatives thereof and wherein the enzymemoiety comprises at least one enzyme of microbial origin selected fromthe group consisting of neutral protease, acid protease, alkalineprotease, lipase, cellulase, dextranase, amylase and asparaginase.

2. Product of claim 1, wherein the polymer is formed by polymerizationof polymerizable acids or anhydrides, or by copolymerizing apolymerizable acid or anhydride with another copolymerizable monomer.

3. Product of claim 1, wherein the polymer is an EMA-type polymer.

4. Product of claim 3, wherein the polymer is EMA.

5. Product of claim 1, which is water-soluble EMA-lipase.

6. Product of claim'l, which is water-soluble EMA-cellulase.

7. Product of claim 1, which is water-soluble EMA- asparaginase.

8. Product of claim 1, which is water-soluble EMA-alkaline protease.

9. Product of claim 1, which is water-soluble EMA-neutral protease.

10. Product of claim 1, which is water soluble EMA- amylase.

11. Product of claim 1, wherein a plurality of different enzymes ofmicrobiological origin are present in the water-soluble polymer-enzymeproduct.

12. Product of claim 1, which is a water-soluble EMA-alkaline protease,neutral protease product.

13. Product of claim 1, which'is a water-soluble EMA-alkaline protease,neutral protease and amylase product.

14. Product of claim 1, which is a water-soluble EMA-alkaline protease,neutral protease and lipase product.

15. Product of claim 12, wherein the ratio of alkaline protease activityto neutral protease activity is about 0.25 to about 1.2 to l.

lb. Product of claim 1, which is water-soluble EMA- dextranase.

17. Product of claim 1, which is a water-soluble EMA- dextranase,neutral protease product.

18. Method of producing a water-soluble enzymatically activepolymer-enzyme product wherein the enzyme is bound covalently throughgroups which are nonessential for enzymatic activity to (a) awater-soluble polymer comprising chains of carboxylic acid or carboxylicacid anhydride units, or (b) a water-soluble polymer comprising units ofcarboxylic acid or carboxylic acid anhydride groups separated by carbonchains of at least one and not more than four carbon atoms, said carbonchains being part of a unit which contains a maximum of 18 carbon atoms,which comprises the step of reacting the polymer and an enzyme undersubstantially noncrosslinking conditions and according to conditionswhich do not operate to destroy enzymatic activity to produce a desiredenzymatically active water-soluble polymer-enzyme product, wherein thepolymer and enzyme are reacted together in dilute aqueous solution orsuspension or with the enzyme in sufficient concentration to favorproduction of a noncrosslinked polymer-enzyme product, and wherein thepolymer is selected from the group consisting of an ethylene/maleicanhydride copolymer, styrene/maleic anhydride copolymer, vinyl methylether/maleic anhydride copolymer, vinyl acetate/maleic anhydridecopolymer, divinyl ether/maleic anhydride cyclocopolymer, polymaleicanhydride, polyacrylic anhydride, and cationic derivatives thereof andwherein the enzyme moiety comprises at least one enzyme of microbialorigin selected from the group consisting of neutral protease, acidprotease, alkaline protease, lipase, cellulase, dextranase, amylase andasparaginase.

19. Process of claim 18 wherein the polymer is reacted with a pluralityof different enzymes of microbiological origin to produce awater-soluble polymer-enzyme product having a plurality of differentenzymes of microbiological origin bound therein.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 5,625Dated December 7, 1971 Inventor(s) It is certified that error appears inthe above-identified patent and that said Letters Patent are herebycorrected as shown below:

The term of this patent subsequent to October 26, 1988 has beendisclaimed.

(SEAL) Attest:

' C. MARSHALL DANN RUTH C. MASON Commissioner of Patents AttestingOfficer and Trademarks Patent No 3 6Z5 8Z7 Dated December 7, 1971lnventorksj Bernard S. Wildi and Thomas L. Westman It is certified thaterror appears in the above-identified patent F ORM P0-105O (0-69) andthat said Letters Patent are hereby corrected as shown below:

'Col. 2, line 53 "(Sephadex Pharmacia Co.

Page 4, line 20 (Se'phadex [TM] Pharmacia Co.

Col. 2, line 73 "evidences" I Page 5, line 7 evidenced Col. 4, line 54"of coupling" Page 8, line 27 or coupling Col. 5, line '18 "of the tothe" Page 10, line 1 of the enzyme to the C01. 7 line 11"pehnylalkyl-phenyl" Page 1 1, line -6 phenylalkyl-phenyl C01. 9,;linel7"7.25%"

Page 18, line 17 amylase C01. 11, line 38 "RF" Page 22, line 3l R Col.12, line 55 "dialyzed against cold water and dialyze Page 26, line 10dialyzed against cold water, and

lyophilized Col. 13, line 47 "gamma" (Amendment 21 May 1971) microlitersPage 27, line 33 c 01. 15, line 14 I "a weighted" Page 30, line 32. Aweighed Col. 16, line 3 '300 m."

Page 32 line 21 300 ml /1/ USCOMM-DC 60370-P69 Q u.s. GOVIRNMENTPRINTING OFFICE: "I! 0-300-334 Col. Page C01. Page C01. Page Col. PageCol. Page (SEAL) Attest:

Patent No.

Inventor(s) M December 7, 1971 Bernard S. Wildi and Thomas L. Westmanline line line line line line line line line line line line line lines10-11 EDWARD M.FLETCHER,JR. Attesting Officer F ORM PC3-1050 (10-69)should read;

' 03,000 r.p'.m."

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

liphl" "con-trifugation" centrifugation lst row of figures under "pH*"should be in line with other rows.

2nd figure underr' 'Pe sin" should be a 54 and not "15 thus 2nd line(8,000 r.p.m.)

"Dean-StarK" Dean-Stark "polymaleicor" polymaleicor llalongll alone"compounds compositions" compounds, compositions Signed and sealed this,13th day of June 1972.-

ROBERT GO'I'TSCHALK Commissioner of Patents USCOMM-DC 60376-P69 a 11.5.GOVERNMENT nummo omce In! mun-3J4

2. Product of claim 1, wherein the polymer is formed by polymerizationof polymerizable acids or anhydrides, or by copolymerizing apolymerizable acid or anhydride with another copolymerizable monomer. 3.Product of claim 1, wherein the polymer is an EMA-type polymer. 4.Product of claim 3, wherein the polymer is EMA.
 5. Product of claim 1,which is water-soluble EMA-lipase.
 6. Product of claim 1, which iswater-soluble EMA-cellulase.
 7. Product of claim 1, which iswater-soluble EMA-asparaginase.
 8. Product of claim 1, which iswater-soluble EMA-alkaline protease.
 9. Product of claim 1, which iswater-soluble EMA-neutral protease.
 10. Product of claim 1, which iswater soluble EMA-amylase.
 11. Product of claim 1, wherein a pluralityof different enzymes of microbiological origin are present in thewater-soluble polymer-enzyme product.
 12. Product of claim 1, which is awater-soluble EMA-alkaline protease, neutral protease product. 13.Product of claim 1, which is a water-soluble EMA-alkaline protease,neutral protease and amylase product.
 14. Product of claim 1, which is awater-soluble EMA-alkaline protease, neutral protease and lipaseproduct.
 15. Product of claim 12, wherein the ratio of alkaline proteaseactivity to neutral protease activity is about 0.25 to about 1.2 to 1.16. Product of claim 1, which is water-soluble EMA-dextranase. 17.Product of claim 1, which is a water-soluble EMA-dextranase, neutralprotease product.
 18. Method of producing a water-soluble enzymaticallyactive polymer-enzyme product wherein the enzyme is bound covalentlythrough groups which are nonessential for enzymatic activity to (a) awater-soluble polymer comprising chains of carboxylic acid or carboxylicacid anhydride units, or (b) a water-soluble polymer comprising units ofcarboxylic acid or carboxylic acid anhydride groups separated by carbonchains of at least one and not more than four carbon atoms, said carbonchains being part of a unit which contains a maximum of 18 carbon atoms,which comprises the step of reacting the polymer and an enzyme undersubstantially noncross-linking conditions and according to conditionswhich do not operate to destroy enzymatic activity to produce a desiredenzymatically active water-soluble polymer-enzyme product, wherein thepolymer and enzyme are reacted together in dilute aqueous solution orsuspension or with the enzyme in sufficient concentration to favorproduction of a noncrosslinked polymer-enzyme product, and wherein thepolymer is selected from the group consisting of an ethylene/maleicanhydride copolymer, styrene/maleic anhydride copolymer, vinyl methylether/maleic anhydride copolymer, vinyl acetate/maleic anhydridecopolymer, divinyl ether/maleic anhydride cyclocopolymer, polymaleicanhydride, polyacrylic anhydride, and cationic derivatives thereof andwherein the enzyme moiety comprises at least one enzyme of microbialorigin selected from the group consisting of neutral protease, acidprotease, alkaline protease, lipase, cellulase, dextranase, amylase andasparaginase.
 19. Process of claim 18 wherein the polymer is reactedwith a plurality of different enzymes of microbiological origin toproduce a water-soluble polymer-enzyme product having a plurality ofDifferent enzymes of microbiological origin bound therein.